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John A. Puskas Ph.D. 2/3/2017
Advances in Liquid Biopsy for Solid Tumors
NATURE REVIEWS | CLINICAL ONCOLOGY VOLUME 10 AUGUST 2013
Liquid Biopsy Origin Other Fluid Sources: CSF Urine
Liquid Biopsy Clinical Applications
Adapted from Alix-Panabieres et. al. (2016) Clinical Appilications of Circulating Tumor Cells and Circulating Tumor DNA as Liquid Biopsy . Cancer discovery
Real-time monitoring
of therapy CTC counts (BC)
KRAS mt on ctDNA (CRC)
AR mt on ctDNA (PC)
Screening and early detection of cancer EGFR mt ctDNA and
CTC counts in (NSCLC)
Stratification and
therapeutic intervention HER2 or ER
expression on CTCs (BC)
CTC counts (BC)-Metabreast trial
Therapeutic targets and resistance mechanisms
KRAS mt (CRC) EGFR my (NSCLC)
Lack of ER expression (BC)
AR mt or ARv7 expression (PC)
Risk for metastatic relapse (prognosis) CTC counts in solid tumors (e.g. breast, prostate, colorectal,
lung and bladder cancers
KRAS mt in ctDNA (CRC)
Clinical Applications
Real time liquid biopsy
Non-invasive blood sampling
Personalized Treatment
CTCs and cfDNA
Ann Transl Med. 2014 Nov;2(11):107. doi: 10.3978/j.issn.2305-5839.2014.08.11.
CTCs and Cell-Free DNA Pros and Cons
Liquid Biopsy Platforms Available at/or Coming to Moffitt Cancer Center
Janssen Diagnostics CellSearch®
ANGLE Parsortix
ddPCR
CellTracks AutoPrep Liquid Biopsy CTCs: The CellSearch® System
CellTracks Analyzer II MagNest®
FDA approved for Metastatic Breast, Colorectal and Prostate Cancers
The CellSearch® System Kit Comparison Epithelial (CTC) cells Epcam capture CK+ PE DAPI+ CD45- APC
FITC channel open MCC 50100 Breast Cancer
Melanoma (CMC) cells CD146 capture MEL+ PE CD45- APC CD34- APC MCC 50140 Melanoma
Epithelial (CXC) cells Epcam capture
CK+ FITC DAPI+ CD45- APC
PE channel open MCC 17924 Bladder Cancer
Endothelial (CEC) and Melanoma cells CD146 capture CD105+ PE DAPI+ CD45- APC
The CellSearch® Antibody Characterization
MDA-MB-231 CELLS
Composite CK-PE DAPI CD45-APC HER-2/neu FITC
SK-BR-3 CELLS
Her2/neu
Other Fully Characterized Antibodies: ER, AR, Ki67, PD-L1
The CellSearch® System: Pros and Cons
Pros Cons
FDA approved Strict CTC definition: Epcam+, DAPI+ CK8, 18, 19+ and CD45-
Automated Limited Available Channels (4) RUO Applications Relatively Expensive
Liquid Biopsy CTCs: ANGLE Parsortix PR1 Separates rare circulating cell populations based on size and deformability and is an epitope-independent enrichment method.
ANGLE Parsortix PR1: How it Works.
Int J Cancer. 2016 Jun 15; 138(12): 2894–2904. Published online 2016 Feb 26. doi: 10.1002/ijc.30007
6.5, 8 and 10 microns sizes available
Harvest cells by reversing flow
ANGLE Parsortix PR1: Pros and Cons
Pros Cons No strict phenotypic definition Cell enrichment only Can detect EMT CTCs Not FDA approved, RUO Cells are viable Cell recovery rates ~75% Color channels defined by user Visualization not included Can isolate CTCs and ctDNA from the same sample
Leukocyte contamination still present
NATURE REVIEWS | CLINICAL ONCOLOGY VOLUME 10 AUGUST 2013
Liquid Biopsy: ctDNA Other potential sources: CSF Urine Cystic Pleural
Applications of Digital PCR
• Circulating Mutations – BRAF V600E – EGFR T790M
• Sub clones in tissue • Mutational screening, minimal residual disease,
tumor evolution • Other fluids-
– Urine, CSF, Pleural
Liquid Biopsy: ctDNA, Why is it Important? Fr
eque
ncy
of c
ases
with
det
ecta
ble
ctD
NA
(%)
Mut
ant f
ragm
ents
per
5m
l
ddPCR Principle: Limiting Dilution PCR
Traditional PCR: One fluorescence
measurement
Digital PCR: Thousands of distinct
fluorescence measurements
VS.
https://en.wikipedia.org/wiki/Digital_polymerase_chain_reaction
How the assay works
Liquid Biopsy: ddPCR EGFR T790M assay Process Overview
EGFR
mut
ant
EGFR Wild Type
T790M mutant droplets
Negative EGFR T790M WT
False positives, droplets with
mix of WT and mutant
ddPCR EGFR T790M Assay Data Analysis
ddPCR EGFR T790M Assay: Sensitivity
0.012 0.42
13.7
7.1
2.23 1.39
0
2
4
6
8
10
12
14
16
NTC WT CTR sample 41%
sample 40.5%
sample 40.1%
sample 40.01%
Conc
0.012 0.42
16.6
8.2
2.8 1.44
0
2
4
6
8
10
12
14
16
18
NTC WT CTR sample 51%
sample 50.5%
sample 50.1%
sample 50.01%
conc
0.012 0.42
34.3
17.5
4.7 0.96
0
5
10
15
20
25
30
35
40
NTC WT CTR sample 61%
sample 60.5%
sample 60.1%
sample 60.01%
Conc
Liquid Biopsy: ddPCR Pros and Cons
Pros Cons
Quantitative 50ng sample/well (150ng)
Fast Little or No Multiplexing
Inexpensive Need to know mutation
Sensitivity
The Future of Liquid Biopsy
+ + + + - -
Thanks!
Morsani Molecular Diagnostic Lab Anthony Magliocco MD Carolyn Loret DeMola Gisela Caceres Ph.D. Mike Gruidl Ph.D. Elena Ryzhova Ph.D. Ravi Kothapalli Ph.D. Liang Nong MD
Moffitt Cancer Center Jhanelle Gray MD
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